The goal of plant breeding is to combine in a single variety/hybrid various desirable traits of the parental lines. For field crops, these traits may include resistance to diseases and insects, tolerance to heat and drought, reducing the time to crop maturity, greater yield, and better agronomic quality. With mechanical harvesting of many crops, uniformity of plant characteristics such as germination and stand establishment, growth rate, maturity, and fruit size, is important.
Field crops are bred through techniques that take advantage of the plant's method of pollination. A plant is self-pollinating if pollen from one flower is transferred to the same or another flower of the same plant. A plant is cross-pollinated if the pollen comes from a flower on a different plant.
Plants that have been self-pollinated and selected for type for many generations become homozygous at almost all gene loci and produce a uniform population of true breeding progeny. A cross between two homozygous lines produces a uniform population of hybrid plants that may be heterozygous for many gene loci. A cross of two plants each heterozygous at a number of gene loci will produce a population of hybrid plants that differ genetically and will not be uniform.
Maize plants (Zea mays L.) can be bred by both self-pollination and cross-pollination techniques. Maize has male flowers, located on the tassel, and female flowers, located on the ear, on the same plant. Natural pollination occurs in maize when wind blows pollen from the tassels to the silks that protrude from the tops of the incipient ears.
The development of maize hybrids requires the development of homozygous inbred lines, the crossing of these lines, and the evaluation of the crosses. Pedigree breeding and recurrent selection are two of the breeding methods used to develop inbred lines from populations. Breeding programs combine desirable traits from two or more inbred lines or various broad-based sources into breeding pools from which new inbred lines are developed by selfing and selection of desired phenotypes. The new inbreds are crossed with other inbred lines and the hybrids from these crosses are evaluated to determine which have commercial potential.
Pedigree breeding starts with the crossing of two genotypes, each of which may have one or more desirable characteristics that is lacking in the other or which complement the other. If the two original parents do not provide all of the desired characteristics, other sources can be included in the breeding population. In the pedigree method, superior plants are selfed and selected in successive generations. In the succeeding generations the heterozygous condition gives way to homogeneous lines as a result of self-pollination and selection. Typically in the pedigree method of breeding five or more generations of selfing and selection is practiced. F.sub.1 .fwdarw.F.sub.2 ; F.sub.2 .fwdarw.F.sub.3 ; F.sub.3 .fwdarw.F.sub.4 ; F.sub.4 .fwdarw.F.sub.5, etc.
A hybrid maize variety is the cross of two inbred lines, each of which may have one or more desirable characteristics lacked by the other or which complement the other. The hybrid progeny of the first generation is designated F.sub.1. In the development of hybrids only the F.sub.1 hybrid plants are sought. The F.sub.1 hybrid is more vigorous than its inbred parents. This hybrid vigor, or heterosis, can be manifested in many ways, including increased vegetative growth and increased yield.
The development of a hybrid maize variety involves three steps: (1) the selection of superior plants from various germplasm pools; (2) the selfing of the superior plants for several generations to produce a series of inbred lines, which although different from each other, each breed true and are highly uniform; and (3) crossing the selected inbred lines with unrelated inbred lines to produce the hybrid progeny (F.sub.1). During the inbreeding process the vigor of the lines decreases. Vigor is restored when two unrelated inbred lines are crossed to produce the hybrid progeny (F.sub.1). An important consequence of the homozygosity and homogeniety of the inbred lines is that the hybrid between any two inbreds will always be the same. Once the inbreds that give the best hybrid have been identified, the hybrid seed can be reproduced indefinitely as long as the homogeneity of the inbred parents is maintained.
A single cross hybrid is produced when two inbred lines are crossed to produce the F.sub.1 progeny. A double cross hybrid, is produced from four inbred lines crossed in pairs (A.times.B and C.times.D) and then the two F.sub.1 hybrids are crossed again (A.times.B).times.(C.times.D). Much of the hybrid vigor exhibited by F.sub.1 hybrids is lost in the next generation (F.sub.2). Consequently, seed from hybrid varieties is not used for planting stock. Likewise, it is very important in the production of hybrid seed to avoid self-pollination and the production and sale of inbred seed to end users.
Hybrid maize seed can be produced by manual detasseling. Alternate strips of two inbred varieties of maize are planted in a field, and the pollen-bearing tassels are removed from one of the inbreds (female). Providing that there is sufficient isolation from sources of foreign maize pollen, the ears of the detasseled inbred will be fertilized only with pollen from the other inbred (male), and the resulting seed is therefore hybrid and will form hybrid plants. Unfortunately, the manual detasseling process is not entirely reliable. Occasionally a female plant will be blown over by a storm and escape detasseling. Or, a detasseler will not completely remove the tassel of the plant. In either event, the female plant will successfully shed pollen and some female plants will be self-pollinated. This will result in seed of the female inbred being harvested along with the hybrid seed which is normally produced.
Alternatively, the female inbred can be mechanically detasseled. Mechanical detasseling is approximately as reliable as manual detasseling, but is faster and less costly. However, most detasseling machines produce more damage to the plants than manual detasseling. Thus, no form of detasseling is presently entirely satisfactory, and a need continues to exist for alternatives which further reduce production costs and the eliminate self-pollination in the production of hybrid seed.
The laborious detasseling process can be avoided by using cytoplasmic male-sterile (CMS) inbreds. Plants of a CMS inbred are male sterile as a result of cytoplasmic factors resulting from the cytoplasmic, as opposed to the nuclear, genome. Thus, this characteristic is inherited exclusively through the female parent, since only the female provides cytoplasm to the fertilized seed. CMS plants are fertilized with pollen from another inbred that is not male-sterile. Pollen from the second inbred may or may not contribute genes that make the hybrid plants male-fertile. Usually seed from detasseled normal maize and CMS produced seed of the same hybrid must be blended to insure that adequate pollen loads are available for fertilization when the hybrid plants are grown.
There can be other drawbacks to CMS. One is an historically observed association of a specific variant of CMS with susceptibility to certain crop diseases. This problem has led to virtual abandonment of use of that CMS variant in producing hybrid maize. In addition, CMS sometimes has a negative association with agronomic performance, particularly in the areas of stalk quality, early seedling vigor, and yield. Finally, CMS exhibits on occasion the potential for breakdown of sterility in certain environments, rendering CMS lines unreliable for hybrid seed production.
Another form of sterility, genic male sterility, is disclosed in U.S. Pat. Nos. 4,654,465 and 4,727,219 to Brat et al. However, this form of genetic male sterility requires maintenance of multiple mutant genes at separate locations within the genome and requires a complex marker system to track the genes and make use of the system convenient.
In self-pollinated species, such as soybeans and cotton, the male and female organs are anatomically juxtaposed. During natural pollination, pollen from the male reproductive organs of a given flower pollinate the female reproductive organs of the same flower. This is in contrast to cross-pollinated species, such as maize, where pollen from the tassel of one plant typically pollinates the silks of another plant through wind dispersal. This can readily occur because of the separation of the male and female reproductive organs. Hybrid production among self-pollinated crops can be difficult because of the close association of the male and female reproductive organs. In addition to the physical difficulty in effecting hybrid production in a self-pollinating crop, the amount of heterosis exhibited in a hybrid is often too low to justify the additional expense required to produce hybrid seed. A reliable form of male sterility would offer the opportunity for improved hybrid plant breeding and increased yields in these species.
Scientists have endeavored to understand development of pollen and the process of fertilization in maize and other plants. Fertilization begins with the germination of mature pollen on a stigmatic surface and the production of a tube which penetrates through the styler tissue. In angiosperms, the growing pollen tube is a conduit for transporting the two sperm cells to the embryo sac where they fuse with the egg and central cells to form the zygote and endosperm, respectively (E. G. Cutter, 1978, Plant Anatomy, Part 1, Experimentation and Interpretation, E. Arnold, Eds., Addison Wesley, London, Chap. 6). Pollen development takes place within the anther and at maturity each grain is a multi-celled spore containing products of both sporophytic gene expression, arising from the inner layer of the anther wall (tapetum), and haploid gene expression from the vegetative cell within each grain (J. P. Mascarenhas, 1990, Annu. Rev. Plant Physiol. Plant Mol Biol. 41:317; J. P. Mascarenhas, 1989, Plant Cell 1:657). Although the process of microsporogenesis is well documented histologically, little is known of the molecular and biochemical factors that are involved in post-dispersal pollen function.
Flavonoids are an abundant class of small molecular weight (-300) plant-specific metabolites which share a common 15 carbon skeletal structure. Modification of the basic structure yields an extensive array of compounds that are classified by the oxidation state and substitution pattern of the various rings. Some classes are pigments (e.g., anthocyanins, chalcones, and particular flavonols and flavones) while other classes are colorless ultraviolet-absorbing compounds. The anthocyanins, particularly pelargonin, cyanidin, and delphinidin, are responsible for the red, blue, and violet plant colors. Other pigmented flavonoids, the chalcones, and some flavonols and flavones are yellow and contribute significantly to the yellow, ivory and cream colored flowers. Pollen flavonoids have been identified in several species where they impact a distinctive yellow color to pollen and can account for a large percentage (2%-5%) of the dry weight (R. Zerbak, M. Bokel, H. Geiger, D. Hess, 1989, Phytochemistry 28;897; R. Wierinann and K. Vieth, 1983 Protoplasma 118;230). There is evidence that the pollen grain is a special environment for flavonoid biosynthesis and/or accumulation as several plant species have unique types of flavonoids in their pollen (O. Ceska and E. D. Styles, 1984, Phytochemistry 23:1822).
Plants having modified flavonoid pigmentation have been previously reported in the literature. For example, a maize mutant producing non-functional white rather than yellow pollen has been previously isolated and characterized (Coe E. H., McCormick S. M. and Modena S. A., 1981, "White Pollen in Maize," J Hered 72:318-320). The white pollen mutant sheds normal amounts of non-pigmented pollen which germinates on the silk, but no seed is set after most pollinations. The condition is sporophytically determined by the expression of stable recessive mutations at the two chalcone synthase (CHS) genes in maize, C2 and Whp. Recently, Agrobacterium-mediated introduction of a CHS transgene into a pigmented inbred petunia stock was reported to suppress the expression of the endogenous CHS gene(s), resulting in flower corollas completely lacking flavonoid pigmentation (Napoli C., Lemieux C. and Jorgensen R., 1990, "Introduction of a Chimeric Chalcone Synthase Gene Into Petunia Results in Reversible Co-repression of Homologous Genes in Trans," Plant Cell 2:279-289). CHS transgene is also suppressed in these plants, and the term co-suppression has been used to describe this phenomenon (Jorgensen R., 1990, "Altered Gene Expression in Plants Due to Trans Interactions Between Homologous Genes," Trends Biotech 8:340-344). The integrated transgene acts like an unlinked dominant inhibitor of the endogenous CHS gene(s) and leads to a complete block in the production of visible flavonoid pigments not only in flower petals but also reproductive organs.
Blockage of CHS gene expression not only results in flavonoid pigmentation deficiencies, but also in plants that are not fertile (Coe, et al., 1981; Taylor, et al., 1992, "Conditional Male Fertility in Chalcone Synthase Deficient Petunia", J. Hered., 83:11-17). It would be highly desirable to be able to control fertility in a manner that plants may be effectively rendered male sterile or fertile as desired.